Human parainfluenza viruses are significant causes of severe respiratory tract disease in infants and young children. HPIV1 is an enveloped, non-segmented, single-stranded, negative-sense RNA virus belonging to the subfamily Paramyxovirinae within the Paramyxoviridae family, which also includes the HPIV2 and HPIV3 serotypes. These serotypes can be further classified as belonging to either the Respirovirus (HPIV1 and HPIV3) or Rubulavirus (HPIV2) genus and are immunologically distinct in that primary infection does not result in cross-neutralization or cross-protection. The HPIV1 genome encodes three nucleocapsid-associated proteins including the nucleocapsid protein (N), the phosphoprotein (P) and the large polymerase (L) and three envelope-associated proteins including the internal matrix protein (M) and the fusion (F) and hemagglutinin-neuraminidase (HN) transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and are the major viral protective antigens. The HPIVs cause respiratory tract disease ranging from mild illness, including rhinitis, pharyngitis, and otitis media, to severe disease, including croup, bronchiolitis, and pneumonia. HPIV1, HPIV2 and HPIV3 have been identified as the etiologic agents responsible for 6.0%, 3.3% and 11.5%, respectively, of hospitalizations of infants and young children for respiratory tract disease. Together these viruses account for approximately 18% of all pediatric hospitalizations due to respiratory disease. A licensed vaccine is currently not available for any of the HPIVs. The major goals of this project are to develop live attenuated virus vaccines that are effective in infants and children against HPIV1, HPIV2, and HPIV3. Pre-clinical research is currently not being conducted with HPIV3 in LID since we have multiple HPIV3 vaccines in clinical trials and three additional HPIV3 vaccine candidates are being prepared for manufacture. Thus, this report focuses on HPIV1 and HPIV2. HPIV1 vaccine development: A novel recombinant human parainfluenza virus type 1 (rHPIV1), rHPIV1-C+P, was generated in which the overlapping open reading frames of the C and P genes were separated in order to introduce mutations into the C gene without affecting P. Infectious rHPIV1-C+P was readily recovered and replicated as efficiently as HPIV1 wild-type (wt) in vitro and in African green monkeys (AGMs). rHPIV1-C+P expressed increased levels of C protein and, surprisingly, activated the type I IFN and apoptosis responses more strongly than HPIV1 wt. rHPIV1-C+P provided a useful backbone for recovering an attenuated P/C gene deletion mutation, (del84-85), which was previously unrecoverable, likely due to detrimental effects of the deletion on the function of the P protein. rHPIV1-C(del84-85)+P and an additional mutant, rHPIV1-C(del169-170) +P, were found to replicate to similar titers in vitro and to activate the type I IFN and apoptosis responses to a similar degree as rHPIV1-C+P. rHPIV1-C(del84-85)+P was highly attenuated in AGMs, and all of the viruses were immunogenic and effective in protecting AGMs against challenge with HPIV1 wt. rHPIV1-C(del84-85)+P will be investigated as a potential live-attenuated vaccine candidate for HPIV1. We further sought to understand the role of the C proteins in apoptosis. Recombinant HPIV1 (rHPIV1) was modified to create rHPIV1-P(C-), a virus in which expression of the C proteins (C, C, Y1 and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C-) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. rHPIV1-P(C-) replicated in vitro as efficiently as HPIV1 wt early during infection, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect (cpe) not observed with rHPIV1 wt which was the result of the activation of apoptosis in rHPIV1-P(C-) infected cells. The apoptosis was initiated by activation of both the intrinsic (caspase 9) and extrinsic (caspase 8) pathways, but was found not to be the cause of the reduction in viral replication. HPIV2 vaccine development: In wild-type human parainfluenza virus type 2 (wt HPIV2), one gene (the P/V gene) encodes both the polymerase-associated phosphoprotein (P) and the accessory cysteine-rich V protein. The P and V proteins share the same N-terminal sequence but have different C-terminal domains due to RNA editing. V is an accessory protein that, in model paramyxoviruses, inhibits the cellular interferon (IFN) response and regulates viral RNA synthesis, in addition to performing other functions that remain less well defined. We generated an HPIV2 virus (rHPIV2-Vko) in which the P/V gene encodes only the P protein to examine the role of V in replication in vivo and for use as a potential live attenuated virus vaccine. Preventing expression of the V protein severely impaired virus recovery from cDNA and growth in vitro, particularly in IFN-competent cells. The rHPIV2-Vko virus, unlike wt HPIV2, strongly induced type I IFN and permitted signaling through the IFN receptor, leading to establishment of a robust antiviral state. rHPIV2-Vko infection induced extensive syncytia and caused dramatic cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-Vko was highly restricted in the upper and lower respiratory tract of African green monkeys and was not detected in differentiated primary human airway epithelial (HAE) cultures, suggesting that the V protein is essential for efficient replication of HPIV2 in vivo and in HAE cultures in vitro. The high degree of restriction of rHPIV2-Vko in non-human primates and in primary HAE cultures suggests that this mutant is over-attenuated and would not be suitable as a live attenuated virus vaccine, but it will be useful to study the function of V during HPIV2 infection. We next characterized wild-type HPIV2 (rHPIV2-WT) infection in a well-established in vitro model of human airway epithelium (HAE) and revealed that the virus replicates to high titer, is shed only apically, targets ciliated cells, and induces minimal cytopathology. Since HPIV2 mutants are currently being developed as live attenuated vaccine candidates, we next sought to determine if infection of HAE with the HPIV2 vaccine candidate, which was described in last years annual report and which is currently in clinical trials, reflects replication in non-human primates. An experimental HPIV2 vaccine strain, rHPIV2-V94(15C)/948L/del1724, that contains both temperature sensitive (ts) and non-ts attenuating mutations, was previously found to be restricted in replication in the upper (URT) and lower respiratory tract (LRT) of African green monkeys (AGMS) and to be protective against wild-type HPIV2 challenge. rHPIV2-V94(15C)/948L/del1724 was reduced in replication by more than 30-fold compared to rHPIV2-WT in HAE cultures at 32C and exhibited little productive replication in cultures at 37C, reflecting a similar restriction of replication in the cooler URT and warmer LRT of AGMs . These data indicate that the HAE model provides a convenient experimental system for examining the cell tropism, cytotoxicity, and attenuation phenotypes of HPIV2 vaccine candidates as well as for characterizing the innate host responses of human airway epithelial tissues to infection. Since clinical trials are the only true tests of vaccine safety and efficacy, the results from this study encourage continued evaluation of rHPIV2-VAC in clinical trials.